Advances in immunotherapy for treatment of lung cancer

Different approaches for treating lung cancer have been developed over time, including chemotherapy, radiotherapy and targeted therapies against activating mutations. Lately, better understanding of the role of the immunological system in tumor control has opened multiple doors to implement different strategies to enhance immune response against cancer cells. It is known that tumor cells elude immune response by several mechanisms. The development of monoclonal antibodies against the checkpoint inhibitor programmed cell death protein 1 (PD-1) and its ligand (PD-L1), on T cells, has led to high activity in cancer patients with long lasting responses. Nivolumab, an anti PD-1 inhibitor, has been recently approved for the treatment of squamous cell lung cancer patients, given the survival advantage demonstrated in a phase III trial. Pembrolizumab, another anti PD-1 antibody, has received FDA breakthrough therapy designation for treatment of non-small cell lung cancer (NSCLC), supported by data from a phase I trial. Clinical trials with anti PD-1/PD-L1 antibodies in NSCLC have demonstrated very good tolerability and activity, with response rates around 20% and a median duration of response of 18 months.

are expressed in tumor cells and antigen presenting cells. Up to 57% of non-small cell lung cancers (NSCLCs) express PD-L1 constitutively or as an acquired adaptive mechanism of immune resistance. Expression of tumor PD-L1 protein in NSCLC was associated with increased local lymphocytic infiltration and longer overall survival (OS) 5 . PD-1 acts within the tumor microenvironment peripherally 6 , inhibiting T-cell signaling, cytotoxic activity, proliferation, survival and effector function of T cells and also promoting differentiation of CD4 + T cells into T regulatory cells and inducing T cell apoptosis 6 .
In view of these immunosuppressive mechanisms, clinical research in the last decade has encompassed targeting these immune check points using monoclonal antibodies like ipilimumab, an anti CTLA-4, and pembrolizumab and nivolumab against PD-1 7,8 . These promising monoclonal antibodies were initially approved for metastatic melanoma and now several PD-1/PD-L1 inhibitors have recently been approved by the FDA also for lung cancer.
Antigen presenting cells present peptides to tumor activated specific T lymphocytes through the major histocompatibility complex class I 9,10 . Cancer cells also augment secretion of gamma interferon (INF-γ) and tumor necrosis factor α (TNF-α) by CD4 + T helper lymphocytes (TH) 10 . High intratumoral T cell density is required to eliminate cancer cells. Prognosis can be affected by the quantity, localization, and phenotype of infiltrating T cells 11 ; high infiltration by cytotoxic T cells confers good prognosis 12 . It is also important to consider that some other inhibitory cells infiltrate tumors, like regulatory T CD4 + cells FOXP3 + (Tregs), cancer associated fibroblasts, myeloid derived suppressor cells and tumor associated macrophages 13 . These cells generate an immunosuppressive microenvironment by several mechanisms, such as TGF-β and interleukin-10 secretion, secretion of platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Tumor cells, on the other hand, produce down-regulation of the major histocompatibility complex class I (MHC-I) and antigen expression and increase PD-L1 expression in tissue. As a result, solid tumors attain an immunological response insufficient to eliminate cancer cells, which is the reason why enhancing the function and quantity of CTLs may be of clinical benefit 14 .
PD-1 receptor is expressed by activated T cells, NK cells, monocytes and B cells and binds PD-L1 (B7-H1 or CD274) and PD-L2 (B7-DC or CD273). PD-L1 is present in malignant cells as well as antigen presenter cells, myeloid cells, epithelial and lymphoid cells, and represents a constitutive or acquired mechanism of immune resistance 33 . PD-1/PD-L1 inhibit cytotoxic T lymphocyte proliferation, survival and effector activity, induce apoptosis of infiltrative T cells and increase the amount of regulatory T cells in the tumor microenvironment 34 .

Clinical development of anti PD-1 and PD-L1 inhibitors in NSCLC
Currently, there are three PD-1/PD-L1 inhibitors available for lung cancer: nivolumab for squamous NSCLC (approved by FDA March 2015), pembrolizumab for the treatment of NSCLC and MPDL3280A from Roche (both granted breakthrough therapy designation) for NSCLC that has progressed on prior treatment (platinum based chemotherapy or targeted therapy for EGFR or ALK positive patients).
NSCLC was found to express PD-L1 in 27% to 57% of cases either in the cellular membrane or the cytoplasm 35,36 . PD-L1 and other molecules that act as immune checkpoints are upregulated in response to substances secreted by lung tumor cells, such as the enzyme IDO which also down-regulates MHC-1 37 . Other immunosuppressive factors like IL-10 and TGF-β are secreted by tumor cells, editing the tumor microenvironment to attenuate immune response 38 . There are several clinical trials ongoing in lung cancer with different immune checkpoints blockers (Tables 1,2).

Anti PD-1 drugs
Nivolumab is a fully humanized anti-PD-1 monoclonal antibody from Bristol Meyer Squibb (BMS). A phase I trial in 2012 in previously treated NSCLC patients treated with intravenous nivolumab every 2 weeks for 12 cycles at different doses (0.1 to 10.0 mg per kilogram of body weight) demonstrated an overall response rate (ORR) of 18% by RECIST 1.1 criteria. These responses were seen in non-squamous as well as squamous NSCLC (ORR of 12% and 33%, respectively) 7 . Long term follow-up of the phase I trial with nivolumab in 129 previously treated NSCLC patients showed that in 50% of patients who responded, response was already evident by week 8 after starting treatment. In this trial, the ORR was 14% up to 25% and OS was 18% at 3 years. The median duration of response was 17 months. Pneumonitis was observed in 7% of patients, and grade 3-4 toxicities in 14%. The most common side effects were fatigue, diarrhea, and anorexia 63 .
The phase III CheckMate 017 trial reported an OS advantage    (Table 1).
Results from a phase III trial CheckMate 026 comparing nivolumab versus chemotherapy in the first line setting for PD-L1 positive NSCLC patients are pending (NCT02041533).
There is also an ongoing phase I trial with multiple arms using combinations of nivolumab with chemotherapy, bevacizumab, ipilimumab or erlotinib (CheckMate 012, NCT01454102). Now a phase III trial is planned to test the combination of nivolumab plus ipilimumab vs. chemotherapy in the first line setting (CheckMate 227, NCT02477826).
In small cell lung cancer (SCLC) a phase III trial will test nivolumab vs. topotecan in the second line setting (CheckMate 331, NCT02481830).
Preliminary results of platinum doublets combined with nivolumab have showed similar response rates when compared to chemotherapy alone in the first line setting (ORR: 33%-47%) and OS rate was 86 % at 18 months in the nivolumab combined with carboplatin and taxol arm 49 .
Pembrolizumab (MK-3475) is an anti PD-1 monoclonal antibody from Merck Sharp & Dohme laboratory. Data from a phase Ib trial in previously treated patients showed an ORR of 21% by RECIST1.1 (26% in treatment naïve and 20% in previously treated patients). In treatment naïve patients, median PFS was 27 weeks, and median overall survival was not reached (OS at 6 months was 86%). In previously treated patients, median PFS was 10 weeks and OS 8.2 months. In the pooled population, median PFS was 13 weeks and 6-month PFS rate was 30%; median OS was 8.2 months with a 6-month OS rate of 64%. The drug was well tolerated with grade 2-4 adverse events in 10% of cases, most commonly pneumonitis 54,64 . In view of these results, pembrolizumab (formerly known as lambrolizumab) was designated as breakthrough therapy for lung cancer treatment by the FDA.
Currently pembrolizumab is being compared with docetaxel in a phase II/III trial in advanced PD-L1 positive NSCLC (NCT01905657). Pembrolizumab is administered every 3 weeks at two different doses (2 mg/kg i.v. every 3 weeks and 10 mg/kg i.v. every 3 weeks) vs. docetaxel (75 mg/m i.v. every 3 weeks).
A phase I trial in PD-L1 positive NSCLC with pembrolizumab (NCT02039674) is currently ongoing, as is a phase I/II trial with ipilimumab or chemotherapy in combination with pembrolizumab (NCT02039674) and a phase II trial of adjuvant pembrolizumab after chemo-radiotherapy for stage III NSCLC patients (NCT02343952) ( Table 2).
In addition to the above mentioned trials, two phase III trials of pembrolizumab at a fixed dose of 200 mg i.v. every 3 weeks for up to 35 treatments is also being compared in the first line setting vs. platinum based chemotherapy in PD-L1 positive and PD-L1 strong positive NSCLC patients (Keynote 042, NCT02220894 and Keynote 024, NCT02142738) ( Table 2).
Data in SCLC patients were presented in May 2015 as preliminary results of an ongoing multi-cohort, phase Ib study of pembrolizumab in patients with PD-L1+ advanced solid tumors (Keynote 028). The SCLC cohort had an ORR of 35% with durable responses 56 (Table 1).
An ongoing phase II trial is testing pembrolizumab in patients with extensive stage small cell lung cancer after completion of combination chemotherapy (NCT02359019) ( Table 2). Anti PD-L1 drugs BMS-936559 is a fully humanized IgG4 monoclonal antibody against PD-L1. This anti PD-L1 was used in 207 patients with advanced stage solid tumors, 75 of whom had NSCLC. The results of the study showed an ORR close to 10% and stable disease for up to 6 months in 18% patients 58 . However, further development of this drug has been halted by Brystol Meyer Squibb ( Table 1). MEDI4736 (Astra Zeneca) is a monoclonal antibody designed with a mutated FC domain in order to prevent antibody-dependent cell mediated cytotoxicity (ADCC). Preliminary results of a phase I trial in patients with different solid tumor types including NSCLC reported clinical benefit and durable disease control with no dose limiting toxicities or grade 3-4 toxicities 60 . Objective response was seen in 23% of patients with pretreated NSCLC (12 out of 53 evaluable patients) in the phase II trial 60 . Results reported at the 2014 ASCO annual meeting showed the drug was well tolerated at all tested doses. Doses were escalated from 0.1 to 10 mg/kg every 2 weeks, with extension to 15 mg/kg every 3 weeks 65 . Pneumonitis, hyperglycemia and colitis were not reported.
Preliminary results from an ongoing study with 346 patients with solid tumors, of whom 143 had NSCLC, used MEDI4736 at dosages of 10 mg/kg every 2 weeks for 1 year. Only 6% of patients had grade 3-4 drug related serious adverse events. The median treatment duration was 8 weeks, and activity was seen as early as 6 weeks. After finishing active therapy, ORR in NSCLC was 13% 59 .
Adjuvant MEDI4736 after chemo-radiotherapy for unresectable stage III NSCLC is currently being tested in a phase III trial (10 mg/kg i.v. every 2 weeks) (NCT02125461). Safety of doses of 0.1-10 mg/kg every 2 weeks or 15 mg/kg every 3 weeks is also been evaluated in a phase I trial in advanced solid tumors including NSCLC (NCT01693562). The anti CTLA-4 IgG2 monoclonal antibody tremelimumab is also being tested in combination with MEDI4736 in a phase Ib trial in NSCLC. (NCT02000947), and a phase III trial in the first line setting will compare the combination with tremelimumab vs. MEDI4736 as single agent vs. chemotherapy in advanced NSCLC according to PD-L1 status ( Table 2).
For EGFR positive patients with T790 mutation, a phase III trial is comparing AZD9291 as single agent vs. combination with MEDI4736 (NCT02143466). Other ongoing phase I trials are combining small molecules with MEDI4736 (NCT02179671, NCT02143466). There is currently a phase II trial recruiting PD-L1 positive patients who have received at least two prior systemic treatment regimens including one platinum-based chemotherapy regimen to receive MEDI4736 (NCT02087423) ( Table 2).
Atezolizumab MPDL3280A from Hoffman la Roche is also an anti-PD-L1 monoclonal antibody, IgG type with Fc domain engineered modification to avoid ADCC. Preliminary phase I results in previously treated advanced NSCLC patients reported a response rate of 23% in squamous and non-squamous NSCLC, 24-week PFS was 48% and median time to response was 12 weeks 61,66 . Results from the phase II POPLAR study presented at the 2015 ASCO annual meeting showed that atezolizumab improved median OS compared with docetaxel in previously treated patients with PD-L1 strong positive NSCLC. For the 47 patients with high PD-L1 expression from 277 included in the study, median OS was not reached in those treated with atezolizumab vs. 11 months in patients treated with docetaxel (HR =0.46; 95% CI: 0.19-1.09). In high expression PD-L1 patients, ORR was 38% with atezolizumab vs. 13% with chemotherapy 62 ( Table 1).
The FIR trial is completed and results are pending. This is a phase II study using this drug in the first setting of PD-L1 positive NSCLC patients (NCT01846416). Other ongoing trials are a phase I study assessing the combination of erlotinib and MPDL3280A in EGFR mutated adenocarcinoma (NCT02013219), a phase III trial where MPDL3280A is being tested at a dose of 1,200 mg i.v. every 3 weeks vs. docetaxel at dose of 75 mg/m 2 i.v. every 3 weeks after chemotherapy failure (NCT02008227), a phase III trial in the first line setting in nonsquamous NSCLC (NCT02409355) and three phase III trials testing the combination of MPDL3280A in the first line setting of non-squamous NSCLC (NCT022367781, NCT02409342, NCT02367794).

Anti CTLA-4 antibodies
Ipilimumab (anti CTL-4 antibody) is a fully humanized Ig G1 monoclonal antibody approved for metastatic melanoma. It was tested in a phase II trial in 334 lung cancer patients (204 NSCLC and 104 SCLC patients) in the first line setting. The trial featured two ipilimumab arms in combination with chemotherapy (paclitaxel plus carboplatin), one arm in a phased schedule and the other one in concomitant schedule vs. a third arm of chemotherapy alone. Median PFS was longer with the phased combination (5.1 vs. 42 months; HR =0.69; 95% CI: 0.48-1.00; P=0.02). Subgroup analysis showed higher activity in squamous cell lung cancer. Toxicity was moderate with grade 3-4 toxic effects in 15% of patients 39 . A phase III trial with ipilimumab in combination with chemotherapy in a phased schedule in squamous NSCLC was completed and results are pending (NCT01285609). Ipilimumab is also being tested in SCLC (NCT01331525, NCT01450761, and NCT02046733) and in combination with ALK and EGFR inhibitors (NCT01998126).
Tremelimumab is a monoclonal antibody similar to ipilimumab and is developed simultaneously. Although tremelimumab is similar to ipilimumab, the pivotal trail in advanced melanoma was negative, and this was the reason why clinical development of the drug was stopped during years. Tremelimumab was also tested in NSCLC in a phase II study including pre-treated patients. Patients were randomized to tremelimumab or best supportive care after four cycles of a platinum combination. The response rate was poor, just 5%, and there were no differences in PFS 41 .

PD-L1 immunohistochemical expression and response to therapy
Immunohistochemistry (IHC) has been used to measure PD-L1 expression in cancer cells, as well as in tumor infiltrating lymphocytes 67 . Interpretation of outcomes is complicated due to the range of techniques and because there are different antibodies used by every pharmaceutical company. Also, timing of the biopsy is a variable that can affect results since expression of the PD-L1 changes during tumor evolution 68,69 . The cut-off at which PD-L1 positivity is considered positive, is an important factor for the interpretation of results. For example, 1% of cut-off has been used in studies with pembrolizumab and depending on this percentage, negativity, light positivity or intense positivity has been defined. Taking into account these different cut-off values, there is a reported 30% of intense positivity of PD-L1 for NSCLC. In the reported studies with nivolumab, a 5% of membrane staining of tumor cells was considered as positive. About 33%-48% of tumor samples were PD-L1 positive in the nivolumab studies. In the studies of MPDL3280A, PD-L1 positivity criteria included 5% of IHC staining on tumor infiltrating lymphocytes and tumor cells. According to these criteria, 25% of NSCLC samples were positive for PD-L1 expression 66 .
A pembrolizumab trial, Keynote 001, has shown improved ORR in patients with positivity for PD-L1 expression. A total of 495 patients were assigned to receive pembrolizumab (at a dose of either 2 or 10 mg per kilogram of body weight every 3 weeks or 10 mg per kilogram every 2 weeks) to either a training group (182 patients) or a validation group (313 patients). Results of PD-L1 expression were reported as the percentage of neoplastic cells with PD-L1 membrane staining; objective responses among all patients was 19.4%, and the median duration of response was 12.5 months. The median PFS was 3.7 months, and median OS was 12.0 months. Cutoff for the training group was PD-L1 expression in at least 50% of tumor cells. A response rate of 45.2% was seen among patients with a proportion score of at least 50% in the validation group, and, among patients with a proportion score of at least 50%; median PFS was 6.3 months while median OS was not reached 68,72 . Other studies as the study from Garon et al. 72 reported higher response rate and longer survival for PD-L1 positive cases.
In other types of tumors like melanoma, the predictive value of PD-L1 can be different, as the response rate in PD-L1 negative cases is higher than in PD-L1 negative lung cancer cases 73 . In melanoma, PD-L1 expression, as well as the presence of infiltrating CD8 lymphocytes, has been described as a better predictive factor for response to anti PD-1 drugs than considering only the PD-L1 expression 74 .

Synergistic combinations and innovative approaches
Synergy can be achieved by targeting different immune checkpoints with combinations, as combining antibodies anti checkpoints with target drugs, antiangiogenic drugs or chemotherapy.
After promising activity in melanoma was reported, trials in lung cancer demonstrated that the combination of a PD-1 inhibitor and anti CTLA-4 antibody was active, with an ORR of 13%-20% 52 . Several ongoing trials are testing different combinations in lung cancer, including several phase III trials ( Table 2).
Interim results from a phase I study evaluating pembrolizumab plus ipilimumab in patients with recurrent NSCLC reported an ORR of 71%, showing a decrease in target lesion burden 57 (Table 1).
Another possible combination is MAPK pathway inhibitors. In the past two decades, targeted therapies in NSCLC, mainly EGFR inhibitors such as erlotinib, gefitinib, afatinib and ALK and ROS-1 inhibitors like crizotinib, have developed rapidly and have shown high response rates 75 . The possible combinations with immunotherapy could presumably allow long lasting effects by means of enhanced tumor antigen presentation by dendritic cells after apoptosis, necrosis and immune checkpoint blockade, allowing infiltrating cytotoxic T cells to attack tumor cells 76 .
Akbay et al. 77 showed that in murine lung tumor cells there is upregulation of tumor PD-L1 via mutant EGFR signaling, and that in preclinical models there was a survival benefit when therapeutic blockade of the PD-L1 pathway was attempted.
A study analyzing biopsies from 123 NSCLC patients reported 56 EGFR mutant and 29 KRAS mutant tumors, with a significant correlation of EGFR mutation with PD-L1 expression, and KRAS mutation with PD-1 expression. Moreover, EGFR positive patients treated with EGFR inhibitors had better survival when they had PD-L1 molecule expression. In a phase I trial, erlotinib plus nivolumab showed a median PFS of 29 months, an OS rate of 64% at 18 months and ORR of 15% 78 .
Trials testing the combination of erlotinib, gefitinib, afatinib or novel anti EGFR drugs plus ipilimumab, nivolumab, MPDL3280A and tremelimumab are currently ongoing. Also a phase I trial is ongoing testing the combination for ALK positive of ceritinib and nivolumab tumors (NCT02393625) ( Table 2).
Several trials are currently assessing different aspects of the combination of antiangiogenic and immunotherapy, including a phase I trial evaluating the safety and tolerability of nivolumab as maintenance therapy in combination with bevacizumab in NSCLC (NCT01454102) ( Table 2).
With regard to combinations with chemotherapy, ipilimumab was studied in a phase III trial with dacarbazine for advanced melanoma. OS was significantly longer in the arm receiving ipilimumab plus dacarbazine than in the group receiving dacarbazine plus placebo (11.2 vs. 9.1 months; HR for death =0.72; P<0.001) 79 . Ipilimumab is currently being studied in combination with lenalidomide for advanced or metastatic cancers with no available standard therapy (NCT01750983). As we have previously mentioned, ipilimumab was studied in a phase II trial in chemotherapy naïve patients with NSCLC. Patients were randomly assigned to receive paclitaxel and carboplatin with either placebo or concurrent ipilimumab or phased ipilimumab. The study met its primary end point of improvement in terms of immune-related progression-free survival (irPFS) for phased ipilimumab vs. the control (HR =0.72; P=0.05), but not for the concurrent arm (HR =0.81; P=0.13) 39 . Nivolumab has also been studied in a phase I trial in combination with carboplatin and nab-paclitaxel in stage IIIB and IV NSCLC (NCT02309177).
There is also a phase I multi-arm study of nivolumab in combination with gemcitabine/cisplatin, pemetrexed/cisplatin, carboplatin/paclitaxel, bevacizumab maintenance, erlotinib, ipilimumab or as monotherapy in subjects with stage IIIB/IV NSCLC (NCT02309177) ( Table 2). Radiation therapy has been described to induce tumor regression in non-irradiated sites, and this rare phenomenon is called the "abscopal effect". There was a case of a melanoma patient treated with ipilimumab and radiotherapy; the patient had a systemic response to localized radiotherapy after progressing on ipilimumab. Non-irradiated areas also regressed and disease remained stable and minimal months later 80 . Radiotherapy has been shown to enhance antigen presentation by myeloid cells within the tumor microenvironment, therefore increasing T-cell killing of the malignant cells, a mechanism which is thought to mediate the abscopal effect. Combinations of radiotherapy with immune checkpoint inhibitors are currently being studied. For example, there is an ongoing phase II trial of ipilimumab with stereotactic body irradiation in advanced lung cancer (NCT02239900), pembrolizumab with hypofractionated stereotactic radiation therapy (NCT02444741), and pembrolizumab with concurrent chemo-radiation for SCLC (NCT02402920).

Toxicity
W hen combining ipilimumab with PD-1 inhibitors like nivolumab in melanoma, drug-related adverse events of grade 3 or 4 were reported in 53% of patients compared with 18% of patients who received ipilimumab monotherapy 72,81,82 . Grade 3 or 4 adverse events, regardless of attribution, were observed in 72% of patients, and grade 3 or 4 treatment-related events were noted in 53%, with the most common events being elevated levels of lipase (in 13% of patients), aspartate aminotransferase (in 13%), and alanine aminotransferase (in 11%). A total of 6 in 28 patients (21%) had grade 3 or 4 treatment-related events that were doselimiting.
Even though ipilimumab is generally well tolerated, severe immune mediated side effects have been observed including enterocolitis, hepatitis, dermatitis, neuropathies, and endocrinopathies. These reactions can manifest during treatment or even months after discontinuation of ipilimumab. In the phase III study of ipilimumab, enterocolitis was the most common severe toxicity, seen in 34 of 511 patients. One percent of patients had bowel perforation and 0.8% died 83,84 . Treatment of severe reactions mainly consists of discontinuation of ipilimumab and initiation of systemic corticosteroids at a dosage of 1 to 2 mg/kg per day of prednisone or equivalent with taper over a period of 1 month once the toxicities have considerably improved 85 .
Nivolumab is a better tolerated drug. The most common adverse reactions (≥20%) reported in a phase III clinical trial were fatigue (50%), dyspnea (38%), musculoskeletal pain (36%), decreased appetite (35%), cough (32%), nausea (29%), and constipation (24%). Only 6% of grade 3 or 4 toxicities are seen. Skin toxicity, including rash, pruritus, and vitiligo are the most commonly seen reactions (31% of the cases). Diarrhea was seen in 11% of cases and pneumonitis in 3%. Transaminitis was seen in 11% and thyroid abnormalities in 3%. Treatment of severe reactions consists of withdrawal of the drug and, if required, prednisone 1 to 2 mg/kg daily should be given until the patient is back at baseline and then tapered over a month 86 .
With pembrolizumab, 13% of patients experienced grade 3 or 4 drug-related adverse events. Common low grade reactions were fatigue, asthenia, fever, chills, myalgias and headaches. Pneumonitis was reported in 4% of cases and grade 3 transaminitis. Toxicities improved after pembrolizumab discontinuation and corticosteroid therapy. Only one case of grade 3 diarrhea was reported, which improved without steroids, and 8% of hypothyroidism which improved with thyroid hormone replacement 72 .

Other immunomodulatory and agonistic molecules
Other immune checkpoint antibodies are currently been studied as single agents, and more interestingly, in combination with other checkpoint inhibitors. The most relevant immune checkpoints currently under clinical investigation are lymphocyte activation gene -3 (LAG-3), KIR, OX 40, GITR, and 4-1BB.
As for LAG-3, preclinical studies have shown co-expression of LAG-3 and PD-1 on tumor infiltrating cells. Combinations of anti-LAG-3 antibodies with anti PD-1 antibodies showed decreased tumor growth and enhanced antitumor immunity, and also maintained immune homeostasis with decreased autoimmune responses 87 . There is currently an ongoing phase I trial testing anti-LAG-3 in advanced NSCLC (NCT01968109).
KIR is a molecule in the NK cell that binds HLA molecules to the surface of tumoral cells, inhibiting NK lymphocyte cytotoxic activity against malignant cells. If KIR is blocked, then NK cells are unblinded and can recognize and attack tumoral cells 88 . Lirilumab is an anti-KIR antibody currently in phase I trials in NSCLC (NCT01750580).
GITR affects regulatory T cells (Tregs) and also acts as a costimulatory receptor expressed after T cell activation that enhances T cell function and survival. Treatment with GITR agonistic antibody destabilizes intra-tumor Tregs allowing for more efficient cytolysis by CD8 + T cells 89 . A phase I trial with anti-GITR antibody TRX-518 is ongoing in advanced solid tumors (NCT01239134). 4-1BB, also known as CD137 receptor, a member of the TNF family of receptors, is an immunomodulatory molecule expressed in immune cells. Urelumab (BMS 663513) is a CD137 agonist that has been tested in a phase I/II study with promising activity, although marked hepatic toxicities have been reported. A study of urelumab in combination with chemoradiotherapy for NSCLC has been terminated (NCT00461110) and the combination with nivolumab is being tested in solid tumors and B cell non-Hodgkin lymphoma (NCT02253992) 90 . There is an ongoing trial with 4-1BB agonist PF-05082566 plus PD-1 inhibitor MK-3475 in patients with solid tumors (NCT02179918) 90 .
OX40 promotes T cell survival and expansion. Preclinical studies showed that OX40 agonists increase anti-tumor immunity and improve disease free survival. Patients treated with one course of the antiOX40 mAb showed regression of at least one metastatic lesion in 12/30 patients and an acceptable toxicity profile 91 .

Cancer vaccines
Two types of vaccines, antigen specific and cell-based, are currently being studied in lung cancer.
MAGE-A3: 35%-42% of NSCLCs have expression of MAGE-A3, which is presented to T cells. A phase II trial showed a correlation between the expression of a gene signature and immune related transcripts associated with better outcome when a MAGE A3 vaccine was used in NSCLC 92 .
L-BLP25 (stimuvax) targets the peptide MUC1 which is overexpressed in lung cancer and associated with poor prognosis 93 . Early trials with L-BLP25 were promising but the phase III trial did not meet its primary endpoint of OS 94 . Two studies are currently ongoing to evaluate L-BLP25, one in combination with bevacizumab for inoperable patients with stage III-IV NSCLC following chemo/radiotherapy (NCT00828009), and another in Asian patients is pending results (NCT01015443). rEGF: 40%-80% of NSCLCs express EGFR. Therefore a vaccine composed of humanized recombinant EGF is being studied. Two trials were started but terminated to initiate a new phase III with a biomarker to enrich the population. Median OS was 11.7 months for patients with anti-EGF antibody response vs. 3.6 months for non-responders 95 . A phase III trial is now planned (NCT01444118).
Anti-telomerase-based vaccine GV1001 showed clinical benefit in a phase I and II trial, with PFS improvement in inoperable NSCLC after chemoradiation (19 months in responders vs. 3.5 months in non-responders, P=0.001). Responders were those who developed GV1001-specific T cell memory responses and had IFN-γ (high)/IL-10(low)/IL-4(low) cytokine profiles 96 .
An NY-ESO-1 vaccine achieved antibody responses in 9 of 10 patients. Of these 10, 2 patients with lung cancer and 1 with esophageal cancer showed stable disease 97 . A clinical trial in cancer patients with tumors expressing NY-ESO-1 tested the vaccine with or without sirolimus (NCT01522820).

Cell based vaccines
Belagenpumatucel-L (Lucanix) is composed of five transforming growth factor B2 (TGF-B2) antisense gene-modified allogenic NSCLC cell lines. In a phase III clinical trial for those patients that were included after completing 12 weeks of chemotherapy, survival analysis showed improvement in overall survival (20.7 vs. 13.4 months, P=0.8) and for those patients treated with radiotherapy, median survival was 40 vs. 10 months (P=0.036) 98 .
Tergenpumatucel-L consists of genetically modified allogeneic NSCLC tumor cells lines with the alpha-(1,3)galactosyltransferase (αGal) moiety on the cell surfaces which generates an innate immune reaction, killing the foreign NSCLC tumor cells. In a phase II clinical trial, 28 patients with metastatic NSCLC or recurrent disease received the vaccine with a median OS of 11.3 months and a long stabilization in 8 of 28 patients with one patient alive after 50 months of follow-up. Patients who received salvage chemotherapy after progressing on tergenpumatucel-L had a better OR to subsequent chemotherapy treatments than patients who had not received prior tergenpumatucel-L 99 .
Peptide vaccines have been also tested in solid tumors, such as the peptide vaccine against the indoleamine 2,3-dioxygenase (IDO) enzyme. In a phase I trial in 15 advanced NSCLC, HLA-A2 positive patients, one patient developed a partial response after 1 year of treatment with the vaccine, and longlasting stable disease was demonstrated in further six patients. Median OS was 25.9 months. Furthermore, expression of IDO was detected in nine of ten tumor biopsies by IHC. In long-term analyses of two clinically responding patients, the ratio of kynurenine/tryptophan in serum (Kyn/Trp) remained stable 100 .

Conclusion
New strategies to treat lung cancer, inducing durable responses to therapy, are currently being developed. The incorporation of immunotherapy to the arsenal of drugs for treatment of lung cancer is a promising approach to reach these goals, with low rates of side effects.
Personalized medicine currently offers the best profile in terms of side effects and efficacy in different cancers, and combination with immune checkpoint blockers could enhance its activity.
In view of promising results reported, now the challenge is finding those biomarkers that can help us select the best treatment approach for every patient.